This invention relates to the preparation of reagents which are useful in the radioimmunoassay (RIA) of digoxin. Specifically, the present invention relates to the reagents formed by the reaction of digoxin and a carboxylic acid in the presence of trifluoroacetic anhydride and then employed in the RIA of digoxin.
Radioimmunoassay is a relatively new type of clinical analysis test. It is now being used to detect a large number of biological agents including digoxin. One of the great advantages of RIA over other clinical diagnostic procedures is the high sensitivity and specificity resulting from the nature of the antigen-antibody or hapten-antibody interactions. Because of the sensitivity, RIA can easily measure antigenic and haptenic concentrations in the ranges of micro-, nano-, and picograms. While all of the conventional principles and techniques of RIA are too extensive to discuss in this application [a good insight into them can be found in an article entitled "Radioimmunoassay" by Skelley et al., Clinical Chemistry, Vol. 19, No. 2, (1973), pp. 146-174], the test is based on the fact that radio-labeled (i.e., labeled with radioactive isotopes) antigen or hapten molecules will compete with nonlabeled antigen or hapten molecules for binding sites on an antibody. Thus, through an RIA analysis, the unknown concentration of nonlabeled antigen or hapten can be found in blood plasma, serum or urine of the patient.
With respect to digoxin, several approaches have been undertaken to radio-label digoxin in order that RIA could be performed. The first approach was to tag the molecule with tritium (.sup.3 H). However, tritium-labeled digoxin has a relatively low specific activity for use in RIA. And, since tritium is a beta emitter and therefore usually counted by use of a liquid scintillation counter, quantifying a specific amount of this material requires laboreous precedures.
.sup.125 I is known as a much better isotope for radio-labeling, but it cannot be chemically bound on the digoxin molecule itself. Workers in this area have made derivatives of the digoxin molecule that accept .sup.125 I either by adding amino acids to the whole molecule [see Wilkinson, S., Chem. Abstr., 80, 105620g (1974)] or by adding amino acids to the steroid part of digoxin [see Rutner et al., Chem. Abstr., 78, 72454j (1973)]. Some of these amino acids contain phenyl groups onto which .sup.125 I can be tagged.
However, the above derivatives presented problems. They usually possess different affinities toward the antibody as compared to the natural digoxin molecule. Moreover, addition of the phenyl group contained in the amino acid would make the derivative bind to other substances besides the antibodies (e.g. proteins in the plasma or serum and the surfaces of plastic test tubes).
Therefore, it was desirable to make a derivative of digoxin having the structure as close as possible to the structure of the natural digoxin molecule, be capable of being radio-labeled by an isotope such as .sup.125 I and possessing similar binding abilities as digoxin.